Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer.

OBJECTIVE: Ovarian cancer, a leading cause of cancer-related deaths in women, remains a formidable challenge, especially in the context of platinum-resistant disease. This study investigated the potential of the benzimidazole derivative BNZ-111 as a novel treatment strategy for platinum-resistant ovarian cancer. METHODS: The human EOC cell lines A2780, HeyA8, SKOV3ip1, A2780-CP20, HeyA8-MDR, and SKOV3-TR were treated with BNZ-111, and cell proliferation, apoptosis, and cell cycle were assessed. RESULTS: It demonstrated strong cytotoxicity in both chemo-sensitive and chemo-resistant epithelial ovarian cancer cell lines, inducing apoptosis and G2/M cell cycle arrest. In vivo experiments using orthotopic and patient-derived xenograft models showed significant tumor growth inhibition without apparent toxicity to vital organs. Unlike paclitaxel, BNZ-111 proved effective in paclitaxel-resistant cells, potentially by bypassing interaction with MDR1 and modulating ß-3 tubulin expression to suppress microtubule dynamics. CONCLUSION: BNZ-111, with favorable drug-like properties, holds promise as a therapeutic option for platinum-resistant ovarian cancer, addressing a critical clinical need in gynecologic oncology.

Synthesis and evaluation of tirbanibulin derivatives: a detailed exploration of the structure-activity relationship for anticancer activity.

Tirbanibulin, an FDA-approved microtubule-targeting agent (MTA) introduced in 2020, represents a pioneering treatment for precancerous actinic keratosis. Despite its failure to gain approval as an anticancer agent due to insufficient efficacy, there remains potential value in extending its application into malignancy treatment through tirbanibulin-based derivatives. Tirbanibulin possesses a distinctive dual mechanism of action involving microtubule and Src inhibition, distinguishing it from other MTAs. In spite of its unique profile, exploration of tirbanibulin's structure-activity relationship (SAR) and the development of its derivatives are significantly limited in the current literature. This study addresses this gap by synthesizing various tirbanibulin derivatives and exploring their SAR through modifications in the core amide motif and the eastern benzylamine part. Our results underscore the critical role of the pyridinyl acetamide core structure for optimal cellular potency, with favorable tolerance observed for modifications at the para position of the benzylamine moiety. Particularly noteworthy is the analogue modified with p-fluorine benzylamine, which exhibited favorable in vivo PK profiles. These findings provide crucial insights into the potential advancement of tirbanibulin-based compounds as promising anticancer agents.

A screening study of high affinity peptide as molecular binder for AXL, tyrosine kinase receptor involving in Zika virus entry.

The recent extensive spread of Zika virus has led to increased interest in the development of early diagnostic tests. To the best of our knowledge, this is the first study to demonstrate the successful use of phage display to identify affinity peptides for quantitative analysis of AXL, a tyrosine kinase receptor involved in Zika virus entry. Biopanning of M13 phage library successfully identified a high affinity peptide, with the sequence AHNHTPIKQKYL. To study the feasibility of using free peptides for molecular recognition, we synthesized a series of amino acid-substituted peptides and examined their binding affinity for AXL using electrochemical impedance spectroscopy and square wave voltammetry. Most synthetic peptides had non-identical random coil structures based on circular dichroism spectroscopy. Of the peptides tested, AXL BP1 exhibited nanomolar binding affinity for AXL. To verify whether AXL BP1 could be used as a peptide inhibitor at the cellular level, two functional tests were carried out: a WST assay for cell viability and qRT-PCR for quantification of RNA levels in Zika virus-infected Huh7 cells. The results showed that AXL BP1 had low cytotoxicity and could block Zika virus entry. These results indicate that newly identified affinity peptides could potentially be used for the development of Zika virus entry inhibitors.

Purification of Boron Nitride Nanotubes Enhances Biological Application Properties.

Commercially available boron nitride nanotubes (BNNTs) and their purified form (pBNNTs) were dispersed in aqueous solutions with various dispersants, and their cytotoxicity and drug encapsulation capacity were monitored. Our data suggest that pBNNTs showed an average increase in dispersibility of 37.3% in aqueous solution in the presence of 10 different dispersants. In addition, 100 µg of pBNNTs induced an average decrease in cytotoxicity of 27.4% compared to same amount of BNNTs in normal cell lines. The same amount of pBNNTs can encapsulate 10.4-fold more drug (camptothecin) compared to BNNTs. These data suggest that the purification of BNNTs improves several of their properties, which can be applied to biological experiments and are thus essential in the biological application of BNNTs.

Properties of differentiated SH-SY5Y grown on carbon-based materials.

Neural cell differentiation has been extensively studied in two-dimensional (2D) cell culture plates. However, the cellular microenvironment and extracellular matrix (ECM) are much more complex and flat 2D surfaces are hard to mimic in ECM. Carbon nanotubes (CNTs) and graphenes are multidimensional carbon-based nanomaterials and may be able to provide extra dimensions on cell growth and differentiation. To determine the effect of CNTs and graphene surfaces on the growth, gene expression, differentiation and functionality of neuroblastoma to a neural cell, SH-SY5Y cells were grown on a 2D (control) surface, a CNT network and a graphene film. The data suggest that SH-SY5Y cells grown on CNT surfaces show an average 20.2% increase in cell viability; 5.7% decrease in the ratio of cells undergoing apoptosis; 78.3, 43.4 and 38.1% increases in SOX2, GFAP and NeuN expression, respectively; and a 29.7% increase in mean firing rate on a multi-electrode array. SH-SY5Y cells grown on graphene film show little or no changes in cell properties compared to cells grown in 2D. The data indicate that the three-dimensional (3D) surface of CNTs provides a favorable environment for SH-SY5Y cells to proliferate and differentiate to neurons.

Development and structure-activity relationship study of SHP2 inhibitor containing 3,4,6-trihydroxy-5-oxo-5H-benzo[7]annulene.

SHP2, a non-receptor protein tyrosine phosphatase encoded by PTPN11 gene, plays an important role in the cell growth and proliferation. Activating mutations of SHP2 have been reported as a cause of various human diseases such as solid tumors, leukemia, and Noonan syndrome. The discovery of SHP2 inhibitor can be a potent candidate for the treatment of cancers and SHP2 related human diseases. Several reports on a small molecule targeting SHP2 have published, however, there are limitations on the discovery of SHP2 phosphatase inhibitors due to the polar catalytic site environment. Allosteric inhibitor can be an alternative to catalytic site inhibitors. 3,4,6-Trihydroxy-5-oxo-5H-benzo[7]annulene 1 was obtained as an initial hit with a 0.097 µM of IC50 from high-throughput screening (HTS) study. After the structure-activity relationship (SAR) study, compound 1 still showed the most potent activity against SHP2. Moreover, compound 1 exerted good potency against SHP2 expressing 2D and 3D MDA-MB-468.

Anticancer effect of XAV939 is observed by inhibiting lactose dehydrogenase A in a 3-dimensional culture of colorectal cancer cells.

XAV939, a tankyrase inhibitor, exerts an anticancer effect in 3-dimensional (3D) cultured SW480 cells, however this is not exhibited in 2-dimensional (2D) cultured SW480 cells. In the current study, XAV939 induced a 3.7-fold increase in cellular apoptosis in 3D culture but not in the 2D culture. However, no significant changes were indicated in cell cycle distribution in the 2D or 3D culture. Based on the observation that protein expression, which was associated with the glycolytic pathway, was increased in the 3D culture, the effect of XAV939 on the patterns of glycolytic protein expression was assessed. XAV939 was revealed to decrease lactose dehydrogenase A (LDHA) expression in 3D cultured SW480 cells, but only exerted a small effect in the 2D culture. The coadministration of XAV939 with the LDHA inhibitor FX11 decreased proliferation in 3D cultured SW480 cells compared with the single administration of FX11, while there was no additive effect in the 2D culture. The lactate assay also indicated that XAV939 decreased lactate secretion in the 3D cell culture but not in the 2D culture. These results suggest that XAV939 exerts an anticancer effect through inhibition of LDHA in the 3D culture.

Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Lactobacillus fermentum Using 2D vs. 3D Culture in Colorectal Cancer Cells.

The aim of this study was to investigate the potential anti-cancer effects of probiotic cell-free supernatant (CFS) treatment using Lactobacillusfermentum for colorectal cancer (CRC) in 3D culture systems. Cell viability was assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assays, whereas apoptosis was monitored through RT-qPCR analysis of Bax, Bak, Noxa, and Bid mRNA expressions in addition to flow cytometry analysis of Lactobacillus cell-free supernatant (LCFS) treatment. Our results showed that the anti-cancer effect of LCFS on cell viability was pronouncedly enhanced in 3D-cultured HCT-116 cells, which was linked to the increased level of cleaved caspase 3. Additionally, upregulation of apoptotic marker gene mRNA transcription was dramatically increased in 3D cultured cells compared to 2D systems. In conclusion, this study suggests that LCFS enhances the activation of intrinsic apoptosis in HCT-116 cells and the potential anti-cancer effects of Lactobacilli mixtures in 3D culture systems. All in all, our study highlights the benefits of 3D culture models over 2D culture modeling in studying the anti-cancer effects of probiotics.

Two-Photon Probe for TNF-α. Assessment of the Transmembrane TNF-α Level in Human Colon Tissue by Two-Photon Microscopy.

We developed Pyr1-infliximab: a two-photon probe for TNF-α. Pyr1-infliximab showed absorption maxima at 280 and 438 nm and an emission maximum at 610 nm in an aqueous buffer and effective two-photon action cross-section values of (520-2830) × 10-50 cm4s/photon in RAW 264.7 cells. After this probe was labeled, it was possible to detect Pyr1-infliximab-transmembrane TNF-α complexes in a live cell and to determine the relative proportion of these complexes in human colon tissues. This proportion among healthy, possibly inflamed, and inflamed tissues of patients with ulcerative colitis was found to be 1.0/4.5/10. This probe may find useful applications for selective detection of transmembrane TNF-α in a live cell or tissue, for quantification of inflammation in human colon tissue or of antidrug antibodies in patients who stop responding to anti-TNF therapy, and for monitoring of the response to this therapy.

Long-Term Stability Monitoring of Printed Proteins on Paper-Based Membranes.

Monitoring of long-term stability of proteins on paper-based membranes is important as it is directly related to paper-based sensor fabrication. By using a simple piezo printhead inkjet printer, recombinant proteins and antibodies were printed on paper-based membranes to test their stability and sensitivity under varying lengths of storage and temperature conditions. Our data show that a printed IgG-HRP antibody on simple printing paper maintains >50% functionality up to ∼2 months under 4 and -20 °C storage. Antibodies printed on polyvinylidene difluoride (PVDF) and nitrocellulose showed 5.3 and 9.7% decreases, respectively, in initial signal intensities compared to printing paper. Prostate-specific membrane antigen and tumor necrosis factor alpha recombinant proteins printed on paper-based membranes can be detected by antibodies, and antibody signal intensities can be detected up to 28 days after storage at 4 and -20 °C when printed on PVDF membrane or printing paper. These data suggest that printed proteins on simple printing paper and PVDF membrane can maintain their functionality up to few months when stored at 4 °C or lower and can be potentially applied in paper-based sensor development.

αVß3-Targeted Delivery of Camptothecin-Encapsulated Carbon Nanotube-Cyclic RGD in 2D and 3D Cancer Cell Culture.

Integrin αvß3 is widely expressed in various types of human cancer lines and plays a key role in angiogenesis for tumor growth and metastasis. Delivery of therapeutics to αvß3-expressing tumors can thus be a promising approach for treating cancer. For targeted delivery of anticancer therapeutics to αvß3-expressing tumor cells, cyclic arginylglycylaspartic acid (RGD) peptide was covalently conjugated to the surface of carboxylic acid-functionalized carbon nanotubes (fCNTs), and the topoisomerase I inhibitor camptothecin (CPT) was encapsulated in the fCNTs (CPT@fCNT-RGD). CPT@fCNT-RGD was successfully delivered to αvß3-expressing A375 cells, and compared with nontargeted CPT@fCNT, it provided 3.78- and 3.02-fold increases in the anticancer effect in 2D and 3D culture. Analysis of apoptosis-related gene expression shows that the expression levels of Bax, cleaved caspase-3, and nuclear factor kappa-light-chain-enhancer of activated B cells were significantly increased in A375 cells incubated with CPT@fCNT-RGD compared with those incubated with CPT@fCNT. These results suggest that cyclic RGD-conjugated CNTs encapsulating an anticancer therapeutic can be a promising platform for treating cancer.

Effect of fibroblast co-culture on the proliferation, viability and drug response of colon cancer cells.

Interactions between cancer cells and the surrounding fibroblasts serve an important role in cancer proliferation. Colon cancer co-culture model with colon fibroblasts and two metastatic models with lung and skin fibroblasts were established, and the co-culture effects on colon cancer cell proliferation, apoptosis and drug response were evaluated. Co-culture with CCD-18Co and BJ reduces SW480 cell proliferation by 4.2 and 5.3%, respectively, while WI-38 acts as a positive regulator and increases SW480 cell proliferation by 36%. CCD-18Co and BJ co-culture can also enhance XAV939 potency against SW480 cells by 16.8 and 27.3%; however, WI-38 co-culture reduces the effect of XAV939 by 38.2%. The present results suggest that, depending on fibroblast type, co-culture can have a positive/negative influence on colon cancer growth; therefore, care should be taken when considering fibroblasts as a target for future cancer therapies.

Encapsulation and Enhanced Delivery of Topoisomerase I Inhibitors in Functionalized Carbon Nanotubes.

The topoisomerase I inhibitors SN-38 and camptothecin (CPT) have shown potent anticancer activity, but water insolubility and metabolic instability limits their clinical application. Utilizing carbon nanotubes as a protective shell for water-insoluble SN-38 and CPT while maintaining compatibility with aqueous media via a carboxylic acid-functionalized surface can thus be a strategy to overcome this limitation. Through hydrophobic-hydrophobic interactions, SN-38 and CPT were successfully encapsulated in carboxylic acid functionalized single-walled carbon nanotubes and dispersed in water. The resulting cell proliferation inhibition and drug distribution profile inside the cells suggest that these drug-encapsulated carbon nanotubes can serve as a promising delivery strategy for water-insoluble anticancer drugs.

Multiplexed Anti-Toxoplasma IgG, IgM, and IgA Assay on Plasmonic Gold Chips: towards Making Mass Screening Possible with Dye Test Precision.

Toxoplasmosis is an infection caused by the protozoan parasite Toxoplasma gondii that can lead to severe sequelae in the fetus during pregnancy. Definitive serologic diagnosis of the infection during gestation is made mostly by detecting T. gondii-specific antibodies, including IgG and IgM, individually in a single serum sample by using commercially available kits. The IgA test is used by some laboratories as an additional marker of acute infection. Most of the commercial tests have failed to reach 100% correlation with the reference method, the Sabin-Feldman dye test for the detection of Toxoplasma IgG antibodies. For Toxoplasma IgM and IgA antibodies, there is no reference method and their evaluation is done by comparing the results of one assay to those of another. There is a need for multiplexed assay platforms, as the serological diagnosis of T. gondii infection does not rely on the detection of a single Ig subtype. Here we describe the development of a plasmonic gold chip with vast fluorescence enhancement in the near-infrared region for simultaneous detection of IgG, IgM, and IgA antibodies against T. gondii in an ∼1-µl serum or whole-blood sample. When 168 samples were tested on this platform, IgG antibody detection sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were all 100%. IgM antibody detection achieved 97.6% sensitivity and 96.9% specificity with a 90.9% PPV and a 99.2% NPV. Thus, the nanoscience-based plasmonic gold platform enables a high-performance, low-cost, multiplexed assay requiring ultrasmall blood volumes, paving the way for the implementation of universal screening for toxoplasmosis infection during gestation.

Visible to Near-Infrared Fluorescence Enhanced Cellular Imaging on Plasmonic Gold Chips.

Rapid and sensitive detections of a variety of surface and intracellular proteins, nucleic acids, and other cellular biomarkers are important to elucidating biological signaling pathways and to devising disease diagnostics and therapeutics. Here, sensitive imaging and detection of cellular proteins on fluorescence-enhancing, nanostructured plasmonic gold (pGold) chips is presented. Imaging of fluorescently labeled cellular biomarkers on pGold is enhanced by 2-30-fold in the visible to near infrared (NIR) range of ≈500-900 nm. The high fluorescence enhancement of >700 nm significantly improves the dynamic range and signal/background ratios of NIR imaging, allowing high-performance multicolor imaging in the visible-NIR range using high quantum yield (QY) visible dyes and lower QY NIR fluorophores. Further, multiple cellular proteins of single cells of various cell types can be detected through microarraying of cells, useful for potentially hundreds and thousands different types of cells assayed on a single chip down to small cell numbers. This work suggests a simple, high throughput, high sensitivity, and multiplexed single-cell analysis method on fluorescence enhancing plasmonic substrates in the entire visible to NIR window.

Fluorophore and dye-assisted dispersion of carbon nanotubes in aqueous solution.

DNA short oligo, surfactant, peptides, and polymer-assisted dispersion of single-walled carbon nanotube (SWCNTs) in aqueous solution have been intensively studied. It has been suggested that van der Waals interaction, π-π stacking, and hydrophobic interaction are major factors that account for the SWCNTs dispersion. Fluorophore and dye molecules such as Rhodamine B and fluorescein have both hydrophilic and hydrophobic moieties. These molecules also contain π-conjugated systems that can potentially interact with SWCNTs to induce its dispersion. Through a systematic study, here we show that SWCNTs can be dispersed in aqueous solution in the presence of various fluorophore or dye molecules. However, the ability of a fluorophore or dye molecule to disperse SWCNTs is not correlated with the stability of the fluorophore/dye-SWCNT complex, suggesting that the on-rate of fluorophore/dye binding to SWCNTs may dominate the efficiency of this process. We also examined the uptake of fluorophore molecules by mammalian cells when these molecules formed complexes with SWCNTs. The results can have potential applications in the delivery of poor cell-penetrating fluorophore molecules.